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Fig. 4. Remnant active ezrin after PKC
knockdown localizes near the tight junction region. CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles as described in Fig. 3. (A,B) Confluent monolayers were extracted and the blots were sequentially processed with anti-T567-P ezrin (A) and then reprobed with anti-ezrin (total protein) (B) antibodies. Molecular mass standards: 200, 117, 85, 40 and 31 kDa. (C-M) Cells were transduced with empty lentivirus vector (C,E,J) or with the same vector carrying an anti-PKC shRNA sequence under a pol III promoter (D,F,G-I,K-M). The cells were fixed and processed with anti-ezrin (total protein) antibody (C,D, red), and anti-T567-P (active) ezrin antibody (E,F, green). C-F are X-Y projections of the apical domain as described in Fig. 3. (G-I) CACO-2 C2Bbe cells expressing anti-PKC shRNA were grown on filters for 8 days, fixed, and processed with anti-T567-P ezrin (green, G,I) and anti-ZO-1 (red, H,I) antibodies. Also, parallel monolayers grown on glass coverslips were processed for scanning electron microscopy of the apical surface (SEM) (J,K). The images are shown as X-Z 3D reconstructions of a confocal stack with the apical side up. (L,M) CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles (L) or lentiviral particles expressing anti-PKC shRNA (M). The cells were selected in puromycin and seeded on Transwell filters. After 6 days, the cells were fixed and processed for immunofluorescence with anti-PKA regulatory subunit IIβ (red) and counterstained with DAPI (blue). The arrows indicate cells with apical distribution of PKA regulatory subunit. Scale bars: 10 µm (C-I,L-M); 1 µm (J,K).
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